| Parathion residue is one of the important detecting items of safety in rice. However, routine detection methods for parathion residues of rice (including HPGC etc.) is not fit for rapid & available inspection of parathion residues of numbers of rice. Therefore, we, consulting advanced technology overseas on the study, according as peculiarity of specific reaction between antigen and antibody, preparing anti-parathion antibody, detecting parathion residues of rice with imply, sensitivity and precise ELISA technology, and obtained satisfactory results as follows:1 , Parathion were coupled to carried protein BSA or OVA by means of the "-N=N-" respectively. By purification and identify with scanning of UV-VIS spectra, two kinds of complete antigen were preparing. Therein, parathion-BSA conjugate are acted as immunogen, and parathion-OVA conjugate as coated antigen. The antiserum was collected from the New Zealand rabbits immunized with above immunogen at an appropriate time, and measured with indirect ELISA on microtiter wells coated with parathion-OVA conjugate. The liters of antiserum from one the rabbits immunized with parathion -BSA conjugate are over 1:409600. This antiserum is used as antibody on study.2, The best concentration of the antiserum and the coated antigen were fixed on 1:6000 and 1:25600 by tests on condition, respectively. With inhibitory assay of indirect ELISA in the concentration, the inhibitory reaction is the better liner with the series of the concentration of parathion standard solutions from 39ng/ml to 5000ng/ml in double time. The correlative coefficient of standard inhibitory curve is 0.993,which show specific reaction between antiserum and parathion. Thereby, with inhibitory assay of indirect ELISA, the quantitative methods detecting parathion was set up.3 >. In order to validating the reliability of the methods, following tests were made.(1) Specificity test: Measuring other drugs with the methods, the antiserum cross-reactivity with reducing parathion, methyl-parathion and paraoxon are 100%, 2.5% and 2.5%, respectively. But the antiserum could not react with p-nitrophenol, malathion, fenitrothion and dimethoate.(2) Accuracy test: 5 samples of rice were spiked with parathion standard from 0ng/ml to5000ng/ml in double time, respectively, prepared simply. The results were obtained with the methods in 4 hours, and the recovery is from 81% to 103%, the average recovery is 89.3%. 5 samples of rice were measure with HPGC in other laboratories, and the coincidence rate comparing of the methods and HPGC are from 76% to 100%, the average coincidence rate is 89.8%.(3) Precision test: 5 samples of rice were spiked with parathion standard from Ong/rnl to 5000ng/ml and were measured on 7 test lots, with statistical treatment, the between-assay average percent coefficient of variations is 6.1%. 5 samples of rice were measured by 3 people at the same laboratory, the within-laboratory average percent coefficient of variations is 5.2%.Above tests showed that the method is simple, rapid, sensitive and precise, it fit for determination of parathion residues in rice. The results of looking related date base showed that this method is set up at first time in China. |
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